1. Field of the Invention
The present invention relates to a Protein S composition comprising recombinant Protein S molecules having complex type N-glycoside-linked sugar chains, having a higher binding activity to a receptor for advanced glycation end products (hereinafter referred to as “RAGE”) than native Protein S present in healthy human blood, and having a high ratio of sugar chains in which fucose is not bound to the complex type N-glycoside-linked sugar chains bound to Protein S than native Protein S present in healthy human blood; and a therapeutic use using the composition.
2. Brief Description of the Background Art
Protein S is a glycoprotein isolated from human plasma, having a molecular weight of about 8×104 daltons and being responsible for an anticoagulant activity in plasma (Non-Patent Document 1, and Patent Document 1). It is known that mRNA expression of Protein S is confirmed in the liver, vascular endothelium, megakaryocytes, testis, and brain and the main production organ of the protein is the liver (Non-Patent Document 2).
The protein moiety of mature Protein S secreted into plasma is a multi-domain type single-chain polypeptide consisting of 635 amino acid residues (SEQ ID NO:8) and containing 17 disulfide bonds in the molecule. Protein S is classified as a vitamin K-dependent protein family, similar to blood coagulation factor VII, factor IX, prothrombin, Protein C, and the like. Native Protein S is known to have four types of domain structures in the molecule thereof, specifically, a γ-carboxylglutamic acid (hereinafter referred to as “Gla”) domain, a thrombin sensitive region, four consecutive epidermal growth factor (EGF)-like domains and a sex hormone-binding globulin (SHBG)-like domain from the amino terminal.
It is known that an asparagine-linked sugar chain contained in the native Protein S molecule is bound to three asparagine residues of Asn458, Asn468 and Asn489 in the SHBG-like domain. Further, it is known that Heerlen mutation, a type of congenital Protein S deficiency, has a replacement of Ser460 with Pro, which results in no addition of a sugar chain to the position of Asn458, thereby decreasing a half-life of Protein S in blood (Non-Patent Documents 3 and 4).
Regarding main physiological functions of Protein S, it is known that Protein S has an inhibitory activity of blood coagulation by promoting the degradation of blood coagulation factors since the Protein S functions as a cofactor which enhances an activity of the APC enzyme by about 20-folds by binding to activated protein C (APC), a serine protease present in human plasma. This anticoagulation activity of Protein S is known as APC cofactor activity.
Further, it is known that Protein S has diverse biological activities, such as a phospholipid binding activity (Non-Patent Document 5), a prothrombinase formation inhibitory activity (Non-Patent Documents 6 and 7), a tenase formation inhibitory activity, a complement C4BP binding activity (Non-Patent Documents 8 and 9), a tissue factor pathway inhibitor (TFPI) cofactor activity (Non-Patent Document 10), and a macrophage surface receptor Axl/Mer/Tyro binding activity (Non-Patent Document 11). It is known that the APC cofactor activity and the phospholipid binding activity among the above-mentioned activities are directly affected by γ-carboxylation of glutamic acid (Glu-to-Gla conversion), a type of post-translational modifications occurring in Protein S molecules in a cell, and by conformational changes of the Gla domain due to coordination of calcium ions in the Gla domain. However, little is known about the relationship between sugar chain structures of Protein S and a variety of biological responses in which Protein S is involved, except the above-mentioned example of the Heerlen mutant.
According to the results of analysis of pathological mechanisms at a molecular level, a role of inflammatory mediator molecules which increases in the bodies of patients has been recently become clear in a variety of human diseases where an excessive increase of blood coagulation and inflammation leads to multi-organ failure, such as sepsis or disseminated intravascular coagulation (DIC).
The receptor for advanced glycation end products (hereinafter referred to as “RAGE”) is a membrane-bound protein known for a long period of time, and is known to be expressed mainly on surfaces of the vascular endothelium, and RAGE functions as a receptor of glycated proteins (AGEs) whose level is increased in blood of diabetic patients. Interestingly, the RAGE protein has been recently detected as a soluble protein (hereinafter referred to as “soluble RAGE”) in peripheral blood of sepsis or DIC patients, and it is clear that a concentration of RAGE in blood is elevated concomitant with poor prognosis (Non-Patent Document 12). It is known that such a soluble RAGE is not only produced by cleavage of RAGE expressed on the membrane (hereinafter referred to as “membrane-type RAGE”), but also is extracellularly secreted as soluble RAGE (Non-Patent Document 13). It has been recently demonstrated that soluble RAGE activates lymphocytes as a result of specific binding to a certain type of integrin molecule (Mac-1) being expressed on the surface of lymphocytes, which consequently promotes the secretion of inflammatory cytokines such as tumor necrosis factor α (TNFα) or interleukin-6 (IL-6) (Non-Patent Document 14). In addition, in a lipopolysaccharide (LPS)-treated mouse sepsis model, administration of anti-RAGE neutralizing antibody has shown to result in significant improvements in a survival rate of mice (Non-Patent Document 15).
From these findings, soluble RAGE is recognized as a mediator molecule of inflammation, so it is considered as a target molecule in the development of inflammatory disease therapeutics. On the other hand, it is known that a membrane-type RAGE not only functions as a receptor of glycated proteins (AGEs), but also serves as a receptor of soluble inflammatory mediator molecules such as high mobility group box chromosomal protein (HMGB) family or S100 (soluble in 100% saturated ammonium sulfate) peptide family (Non-Patent Document 16). It is known that a variety of events relating to the enhanced inflammation, such as cellular chemotaxis, the enhanced cellular secretion of inflammatory cytokines, the elevated expression levels of adhesion molecules such as ICAM or VCAM, are introduced by the binding of HMGB or S100 to cells expressing membrane-type RAGE (Non-Patent Documents 17 and 18).
Meanwhile, an anti-inflammatory response of Protein S has not yet been known up to date, and further, there is no finding relating to the interaction between Protein S and RAGE.